Dendritic cells: the host Achille's heel for mucosal pathogens?

Mucosal surfaces represent the main sites of interaction with environmental microorganisms and antigens. Sentinel cells, including epithelial cells and dendritic cells (DCs), continuously sense the environment and coordinate defenses for the protection of mucosal tissues. DCs play a central role in the control of adaptive immune responses owing to their capacity to internalize foreign materials, to migrate into lymph nodes and to present antigens to naive lymphocytes. Some pathogenic microorganisms trigger epithelial responses that result in the recruitment of DCs. These pathogens hijack the recruited DCs to enable them to infect the host, escape the host's defense mechanisms and establish niches at remote sites.

Innate immunogenetics: a tool for exploring new frontiers of host defence.

The discovery of innate immune genes, such as those encoding Toll-like receptors (TLRs), nucleotide-binding oligomerisation domain-like receptors (NLRs), and related signal-transducing molecules, has led to a substantial improvement of our understanding of innate immunity. Recent immunogenetic studies have associated polymorphisms of the genes encoding TLRs, NLRs, and key signal-transducing molecules, such as interleukin-1 receptor-associated kinase 4 (IRAK4), with increased susceptibility to, or outcome of, infectious diseases. With the availability of high-throughput genotyping techniques, it is becoming increasingly evident that analyses of genetic polymorphisms of innate immune genes will further improve our knowledge of the host antimicrobial defence response and help in identifying individuals who are at increased risk of life-threatening infections. This is likely to open new perspectives for the development of diagnostic, predictive, and preventive management strategies to combat infectious diseases.

Macrophage migration inhibitory factor and host innate immune defenses against bacterial sepsis.

Macrophages are essential effector cells of innate immunity that play a pivotal role in the recognition and elimination of invasive microorganisms. Mediators released by activated macrophages orchestrate innate and adaptive immune host responses. The cytokine macrophage migration inhibitory factor (MIF) is an integral mediator of the innate immune system. Monocytes and macrophages constitutively express large amounts of MIF, which is rapidly released after exposure to bacterial toxins and cytokines. MIF exerts potent proinflammatory activities and is an important cytokine of septic shock. Recent investigations of the mechanisms by which MIF regulates innate immune responses to endotoxin and gram-negative bacteria indicate that MIF acts by modulating the expression of Toll-like receptor 4, the signal-transducing molecule of the lipopolysaccharide receptor complex. Given its role in innate immune responses to bacterial infections, MIF is a novel target for therapeutic intervention in patients with septic shock.

Investigation of the hepatitis C virus replication complex.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Early expression of the type III secretion system of Parachlamydia acanthamoebae during a replicative cycle within its natural host cell Acanthamoeba castellanii.

The type three secretion system (T3SS) operons of Chlamydiales bacteria are distributed in different clusters along their chromosomes and are conserved at both the level of sequence and genetic organization. A complete characterization of the temporal expression of multiple T3SS components at the transcriptional and protein levels has been performed in Parachlamydia acanthamoebae, replicating in its natural host cell Acanthamoeba castellanii. The T3SS components were classified in four different temporal clusters depending on their pattern of expression during the early, mid- and late phases of the infectious cycle. The putative T3SS transcription units predicted in Parachlamydia are similar to those described in Chlamydia trachomatis, suggesting that T3SS units of transcriptional expression are highly conserved among Chlamydiales bacteria. The maximal expression and activation of the T3SS of Parachlamydia occurred during the early to mid-phase of the infectious cycle corresponding to a critical phase during which the intracellular bacterium has (1) to evade and/or block the lytic pathway of the amoeba, (2) to differentiate from elementary bodies (EBs) to reticulate bodies (RBs), and (3) to modulate the maturation of its vacuole to create a replicative niche able to sustain efficient bacterial growth.

The separate and combined effects of MHC genotype, parasite clone, and host gender on the course of malaria in mice.

BACKGROUND: The link between host MHC (major histocompatibility complex) genotype and malaria is largely based on correlative data with little or no experimental control of potential confounding factors. We used an experimental mouse model to test for main effects of MHC-haplotypes, MHC heterozygosity, and MHC x parasite clone interactions. We experimentally infected MHC-congenic mice (F2 segregants, homo- and heterozygotes, males and females) with one of two clones of Plasmodium chabaudi and recorded disease progression. RESULTS: We found that MHC haplotype and parasite clone each have a significant influence on the course of the disease, but there was no significant host genotype by parasite genotype interaction. We found no evidence for overdominance nor any other sort of heterozygote advantage or disadvantage. CONCLUSION: When tested under experimental conditions, variation in the MHC can significantly influence the course of malaria. However, MHC heterozygote advantage through overdominance or dominance of resistance cannot be assumed in the case of single-strain infections. Future studies might focus on the interaction between MHC heterozygosity and multiple-clone infections.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify 'plasticity'. In this review, we want to place 'plasticity' in a historical perspective still taking into account ethical and political implications.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Effects of genetic alterations in arbuscular mycorrhizal fungi on plant growth and on their response to a change of host

Abstract :The majority of land plants form the symbiosis with arbuscular mycorrhizal fungi (AMF). The AM symbiosis has existed for hundreds of millions of years but little or no specificity seems to have co- evolved between the partners and only about 200 morphospecies of AMF are known. The fungi supply the plants most notably with phosphate in exchange for carbohydrates. The fungi improve plant growth, protect them against pathogens and herbivores and the symbiosis plays a key role in ecosystem productivity and plant diversity. The fungi are coenocytic, grow clonally and no sexual stage in their life cycle is known. For these reasons, they are presumed ancient asexuals. Evidence suggests that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. Consequently, the nucleotype content of new clonal offspring could potentially be altered by segregation of nuclei at spore formation and by genetic exchange between different AMF. Given the importance of AMF, it is surprising that remarkably little is known about the genetics and genomics of the fungi.The main goal of this thesis was to investigate the combined effects of plant species differences and of genetic exchange and segregation in AMF on the symbiosis. This work showed that single spore progeny can receive a different assortment of nucleotypes compared to their parent and compared to other single spore progeny. This is the first direct evidence that segregation occurs in AMF. We then showed that both genetic exchange and segregation can lead to new progeny that differentially alter plant growth compared to their parents. We also found that genetic exchange and segregation can lead to different development of the fungus during the establishment of the symbiosis. Finally, we found that a shift of host species can differentially alter the phenotypes and genotypes of AMF progeny obtained by genetic exchange and segregation compared to their parents.Overall, this study confirms the multigenomic state of the AMF Glomus intraradices because our findings are possible only if the fungus contains genetically different nuclei. We demonstrated the importance of the processes of genetic exchange and segregation to produce, in a very short time span, new progeny with novel symbiotic effects. Moreover, our results suggest that different host species could affect the fate of different nucleotypes following genetic exchange and segregation in AMF, and can potentially contribute to the maintenance of genetic diversity within AMF individuals. This work brings new insights into understanding how plants and fungi have coevolved and how the genetic diversity in AMF can be maintained. We recommend that the intra-ir1dividual AMF diversity and these processes should be considered in future research on this symbiosis.Résumé :La majorité des plantes terrestres forment des symbioses avec les champignons endomycorhiziens arbusculaires (CEA). Cette symbiose existe depuis plusieurs centaines de millions d'années mais peu ou pas de spécificité semble avoir co-évoluée entre les partenaires et seulement 200 morpho-espèces de CEA sont connues. Le champignon fournit surtout aux plantes du phosphate en échange de carbohydrates. Le champignon augmente la croissance des plantes, les protège contre des pathogènes et herbivores et la symbiose joue un rôle clé dans la productivité des écosystèmes et de la diversité des plantes. Les CEA sont coenocytiques, se reproduisent clonalement et aucune étape sexuée n'est connue dans leur cycle de vie. Pour ces raisons, ils sont présumés comme anciens asexués. Des preuves suggèrent que les CEA ont des populations de nucleotypes différents coexistant dans un cytoplasme commun. Par conséquent, le contenu en nucleotype des nouveaux descendants clonaux pourrait être altéré par la ségrégation des noyaux lors de la fonnation des spores et par l'échange génétique entre différents CEA. Etant donné l'importance des CEA, il est surprenant que si peu soit connu sur la génétique et la génomique du champignon.Le principal but de cette thèse a été d'étudier les effets combinés de différentes espèces de plantes et des mécanismes d'échange génétique et de ségrégation chez les CEA sur la symbiose. Ce travail a montré que chaque nouvelle spore produite pouvait recevoir un assortiment différent de noyaux comparé au parent ou comparé à d'autres nouvelles spores. Ceci est la première preuve directe que la ségrégation peut se produire chez les CEA. Nous avons ensuite montré qu'à la fois l'échange génétique et la ségrégation pouvaient mener à de nouveaux descendants qui altèrent différemment la croissance des plantes, comparé à leurs parents. Nous avons également trouvé que l'échange génétique et la ségrégation pouvaient entraîner des développements différents du champignon pendant l'établissement de la symbiose. Pour finir, nous avons trouvé qu'un changement d'espèce de l'hôte pouvait altérer différemment les phénotypes et génotypes des descendants issus d'échange génétique et de ségrégation, comparé à leurs parents.Globalement, cette étude confirme l'état multigénomique du CEA Glumus intraradices car nous résultats sont possibles seulement si le champignon possède des noyaux génétiquement différents. Nous avons démontrés l'importance des mécanismes d'échange génétique et de ségrégation pour produire en très peu de temps de nouveaux descendants ayant des effets symbiotiques nouveaux. De plus, nos résultats suggèrent que différentes espèces de plantes peuvent agir sur le devenir des nucleotypes après l'échange génétique et la ségrégation chez les CEA, et pourraient contribuer à la maintenance de la diversité génétique au sein d'un même CEA. Ce travail apporte des éléments nouveaux pour comprendre comment les plantes et les champignons ont coévolué et comment la diversité génétique chez les CEA peut être maintenue. Nous recommandons de considérer la diversité génétique intra-individuelle des CEA et ces mécanismes lors de futures recherches sur cette symbiose.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Host specialist clownfishes are environmental niche generalists.

Why generalist and specialist species coexist in nature is a question that has interested evolutionary biologists for a long time. While the coexistence of specialists and generalists exploiting resources on a single ecological dimension has been theoretically and empirically explored, biological systems with multiple resource dimensions (e.g. trophic, ecological) are less well understood. Yet, such systems may provide an alternative to the classical theory of stable evolutionary coexistence of generalist and specialist species on a single resource dimension. We explore such systems and the potential trade-offs between different resource dimensions in clownfishes. All species of this iconic clade are obligate mutualists with sea anemones yet show interspecific variation in anemone host specificity. Moreover, clownfishes developed variable environmental specialization across their distribution. In this study, we test for the existence of a relationship between host-specificity (number of anemones associated with a clownfish species) and environmental-specificity (expressed as the size of the ecological niche breadth across climatic gradients). We find a negative correlation between host range and environmental specificities in temperature, salinity and pH, probably indicating a trade-off between both types of specialization forcing species to specialize only in a single direction. Trade-offs in a multi-dimensional resource space could be a novel way of explaining the coexistence of generalist and specialists.